CN115028706B - 马乳酪蛋白小分子肽在制备二肽基肽酶ⅳ抑制剂中的用途 - Google Patents
马乳酪蛋白小分子肽在制备二肽基肽酶ⅳ抑制剂中的用途 Download PDFInfo
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- CN115028706B CN115028706B CN202210004446.6A CN202210004446A CN115028706B CN 115028706 B CN115028706 B CN 115028706B CN 202210004446 A CN202210004446 A CN 202210004446A CN 115028706 B CN115028706 B CN 115028706B
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- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
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- C07K—PEPTIDES
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种小分子肽的新用途，即马乳酪蛋白小分子肽在制备二肽基肽酶Ⅳ抑制剂中的应用，所述小分子肽的氨基酸序列为Pro‑Gln‑Pro‑Ile‑Glu‑Arg‑Thr，分子量为839.95 Da；本发明小分子肽具有二肽基肽酶Ⅳ抑制活性，对二肽基肽酶Ⅳ的抑制率为86%，半抑制浓度（IC50）为0.726 mg/mL；且能改善高糖诱导的（人肝癌）Hepg2细胞胰岛素抵抗，显著提高Hepg2细胞活性，改善Hepg2细胞的葡萄糖消耗与葡萄糖摄取；本发明小分子肽可应用于制备治疗或辅助治疗2型糖尿病的药物，且制备简单，适用于工业化生产和市场推广应用。
Description
技术领域
本发明属于生物技术领域，具体涉及一种马乳酪蛋白小分子肽在制备二肽基肽酶Ⅳ抑制剂中的应用。
背景技术
糖尿病是胰岛素分泌减少、或胰岛素作用缺陷、或两者同时存在而造成血糖升高、脂肪代谢紊乱等诱发的代谢疾病。糖尿病成为全球关注的健康问题，90％以上的糖尿病患者患有2型糖尿病（Type2 Diabetes Mellitus，T2DM）。目前已有多类降血糖药物，例如DPP-4抑制剂类、α-葡萄糖苷酶抑制剂类、胰高血糖素样肽-1受体激动剂类等，但这些药物长期服用对人体有副作用。因此人们越来越倾向于预防保健和食疗，而来源于食品且无副作用的乳源二肽基肽酶Ⅳ抑制剂、α-葡萄糖苷酶抑制剂等在国内具有巨大的潜在市场，具有副作用小、成本低等优点。乳源中马乳的营养物质含量较高且不含或少含β-乳球蛋白，因此过敏性较低，易消化、吸收和代谢。
发明内容
本发明提供了一种小分子肽的新用途，其来源于马乳酪，该小分子肽的氨基酸序列为Pro-Gln-Pro-Ile-Glu-Arg-Thr，分子量为839.95Da，其具有二肽基肽酶Ⅳ抑制活性。
本发明所述抑制二肽基肽酶Ⅳ活性的成分（或有效成分）为上述小分子肽，还可以加入一种或多种药物上可接受的辅料，如制成胶囊或丸剂、粉剂、片剂、粒剂、口服液和注射液等，即制成药剂学上适宜的使用剂型；可应用于制备治疗或者辅助治疗2型糖尿病的药物。
本发明目的通过如下方案实现：
（1）马乳酪蛋白的获取：鲜马乳在4~10℃、2000~12000g下离心1~15min脱脂，脱脂马乳在20~50℃水浴中用HCl调pH至1.5~8.5，然后2000~12000g离心1~15min，取沉淀用冻干机冷冻干燥，收集冻干粉；
（2）菌种活化：将酵母菌接种至YPD液体培养基中，在25~40℃、50~300rpm下培养6~48h，连续增殖2~4代，接种量为体积百分比0.1~5%，活化后菌液的活菌数不少于108CFU/mL，备用；所述酵母菌为单孢酿酒酵母、乳酸克鲁维酵母、酿酒酵母、毕赤酵母中的一种或几种；
（3）马乳酪蛋白的发酵：将步骤（1）马乳酪蛋白冻干粉用蒸馏水配成溶液后，在60~150℃灭菌1~30min，降至室温后接种活化好的酵母菌，接种量为0.1~5%，进行发酵；发酵条件为20～40℃、摇床转速为50~300rpm、发酵时间为12~100h；
（4）发酵产物的酶解：将发酵液pH调至1~9，添加蛋白酶，蛋白酶的添加量为质量比2~10%，在20~60℃反应0.5~126h；所述蛋白酶为胰蛋白酶、胃蛋白酶、中性蛋白酶、碱性蛋白酶、弹性蛋白酶、菠萝蛋白酶、木瓜蛋白酶、复合蛋白酶、风味蛋白酶等的一种或几种，均为常规市售蛋白酶；
（5）酶解结束后，将发酵酶解液pH调至1.5~9.5，用不同截留分子量的截留超滤膜对溶液进行分离，获得不同组分的液体，用冻干机冷冻干燥，收集冻干粉；
（6）将含有不同组分的冻干粉分别溶于蒸馏水中，制得20~800mg/mL的溶液，检测各溶液的二肽基肽酶Ⅳ抑制活性，选择抑制活性最好的液体进行葡聚糖凝胶柱层析和反相高效液相色谱（RP-HPLC）分离纯化，选择RP-HPLC纯化后具有二肽基肽酶Ⅳ抑制活性的峰进行LC-MS鉴定，鉴定后获得的肽序列在BIOPEP进行二肽基肽酶Ⅳ抑制活性预测，选择预测活性最好的进行固相合成验证，最终获得本发明的小分子肽。
本发明的优点和积极效果如下：
1、本发明证实了马乳酪蛋白小分子肽Pro-Gln-Pro-Ile-Glu-Arg-Thr（PQPIERT）)可显著抑制二肽基肽酶Ⅳ活性，对二肽基肽酶Ⅳ的抑制率为86%，半抑制浓度（IC50）为0.726 mg/mL，为进一步揭示马乳酪蛋白的降血糖机制提供依据；
2、本发明证实了马乳酪蛋白小分子肽PQPIERT改善高糖诱导的Hepg2细胞胰岛素抵抗，显著提高Hepg2细胞活性，改善Hepg2细胞的葡萄糖消耗与葡萄糖摄取；
3、本发明小分子肽可应用于制备治疗或辅助治疗2型糖尿病的药物，具有制备简单、安全无副作用等优点。
附图说明
图1为截留超滤膜分离后不同分子质量组分对二肽基肽酶Ⅳ抑制活性结果；
图2为葡聚糖凝胶层析色谱分离峰示意图；
图3为葡聚糖凝胶层析色谱分离后对应分离峰组分的二肽基肽酶Ⅳ抑制活性检测结果；
图4为采用RP-HPLC分离后分离峰示意图；
图5为RP-HPLC分离后对应分离峰组分的二肽基肽酶Ⅳ抑制活性检测结果；
图6为RP-HPLC二次分离后对应分离峰示意图；
图7为RP-HPLC二次分离后对应分离峰组分的二肽基肽酶Ⅳ抑制活性检测结果；
图8为RP-HPLC三次分离后对应分离峰示意图；
图9为RP-HPLC三次分离后对应分离峰组分的二肽基肽酶Ⅳ抑制活性检测结果；
图10为R2-2-2组分的高效液相色谱图；
图11为小分子肽PQPIERT的质谱鉴定图；
图12为小分子肽PQPIERT的酶抑制动力学实验结果，图中0、0.25、0.5mg/mL为小分子肽浓度；
图13为小分子肽PQPIERT对Hepg2细胞活性的影响，图中Metformin为二甲双胍，NT为阴性对照；
图14为小分子肽PQPIERT对高糖诱导的胰岛素抵抗Hepg2细胞葡萄糖消耗的影响，图中Metformin为二甲双胍，NT为阴性对照，IR为模型组；
图15为小分子肽PQPIERT对高糖诱导的胰岛素抵抗Hepg2细胞葡萄糖摄取的影响，图中Metformin为二甲双胍，NT为阴性对照。
具体实施方式
下面通过实施例对本发明的技术方案作进一步详细说明，但本发明的内容并不局限于此，本实施例中方法如无特殊说明均为常规方法，所用材料、试剂等如无特殊说明均从商业途径所得到；
下述实施例中二肽基肽酶Ⅳ抑制活性检测的方法为：
用pH8.0的100mmol/L Tris-HCL缓冲液将DPP-Ⅳ酶和甘氨酰脯氨酸对硝基苯胺（GPP）分别配置成 0.025U/mL和1.2mmol/L的溶液。移取25μL GPP和25μL样品小分子肽溶液于96孔板中，充分混合，在37℃下孵育10min，然后再加入50μL DPP-Ⅳ粗提液（从兔肠中提取出来含ACE的提取液）启动反应，37℃条件下充分反应60min；加入100μL pH 4.0浓度为1mmoL/L的乙酸-乙酸钠溶液终止反应，酶标仪检测并记录405nm波长下的吸光值，每个样品做三个平行反应；由于反应体系略带浑浊，因此实验设置酶活性组（缓冲液+酶+底物）、酶空白组（缓冲液+底物）、样品组（酶+样品+底物）和样品对照组（缓冲液+样品+底物）。酶活性组只有GPP和DPP-Ⅳ进行反应，等量缓冲液代替小分子肽样品溶液，得到同等反应条件下可生成PNP的最高含量，以此方法比对出小分子肽样品是否具起到抑制作用；酶空白组只添加GPP，其余用等量缓冲液代替，排除GPP在孵育过程中自然分解生成显示物质PNP的干扰；样品对照组同时添加缓冲液、样品小分子肽和底物，排除在孵育过程中样品小分子肽对吸光度的干扰，DPP-Ⅳ酶抑制活性的计算：
抑制率（%）=1-（A样品组−A样品对照组）/（B酶活性组−B酶空白组）×100%
A样品组（DPP-IV+样品+ GPP）：加入小分子肽样品后测定的小分子肽样品和PNP吸光度；
A样品对照组（缓冲液+样品+ GPP）：小分子肽样品的吸光度；
B酶活性组（缓冲液+DPP-IV + GPP）：不加样品时测定的PNP吸光度；
B酶空白组（缓冲液+ GPP）：用缓冲液代替酶测定的PNP吸光度。
实施例1：小分子肽的获取
（1）马乳酪蛋白的获取：鲜马乳在4℃、5000g下离心5min脱脂，脱脂马乳在45℃水浴中用HCl调pH至4.2，10000g离心5min，取沉淀用冻干机冷冻干燥，收集冻干粉；
（2）菌种活化：酵母菌接种至YPD液体培养基中，在30℃、200rpm下培养12h，连续增殖3代，接种量为体积百分比4%，活化后菌液的活菌数不少于108CFU/mL，备用；
（3）马乳酪蛋白的发酵：将步骤（1）马乳酪蛋白冻干粉用蒸馏水配成100μg/mL溶液，在115℃灭菌10min，降温至室温后，接种活化好的单孢酿酒酵母（Kazachstania unispora）KU530（该菌种在文献“发酵和酶解法处理马乳酪蛋白制备ACE抑制肽”公开），接种量为4%，进行发酵；发酵条件为：30℃、摇床转速为200rpm、发酵时间为72 h；
（4）发酵产物的酶解：将发酵液pH调至7.75，添加胰蛋白酶，胰蛋白酶的添加量为6.5%，在37℃反应1.5 h；
（5）酶解结束后，发酵酶解液pH调至8.3，用截留量为10kDa及3kDa的截留超滤膜过滤，得到含有分子量>10kDa、3-10kDa、<3kDa组分的溶液，分别冷冻干燥，收集冻干粉；冻干粉溶于蒸馏水制得100mg/mL的溶液，测各组分二肽基肽酶Ⅳ抑制活性（图1）；选取抑制活性最好的含有分子量<3kDa组分，然后将该组分的溶液用葡聚糖凝胶SephedaxG-10层析，用0.5mL/min的蒸馏水洗脱，280nm监测洗脱液的吸光度值变化，收集分离峰对应的液体S1、S2、S3（图2），分别冷冻干燥，冻干粉用蒸馏水配制成浓度100mg/mL的溶液，并测定各组分对二肽基肽酶Ⅳ抑制活性（图3）；
（6）选择抑制活性最好的S1利用半制备RP-HPLC进行分离（图4），收集4个组分液体并旋蒸，测定不同组分对二肽基肽酶Ⅳ抑制活性（图5）；选择抑制活性最好的R2经RP-HPLC分离得到3个组分（图6），测定不同组分对二肽基肽酶Ⅳ抑制活性（图7）；选择抑制活性最好的R2-2经RP-HPLC分离得到3个组分（图8）和对应的组分对二肽基肽酶Ⅳ抑制活性（图9）；选择抑制活性最好的R2-2-2经RP-HPLC分离分离得到1个组分（图10）
（7）将R2-2-2峰组分进行LC-MS鉴定，分析时长：35min。检测方式：正离子。小分子肽和小分子肽的碎片的质量电荷比按照下列方法采集：每次全扫描（full scan）后采集10个碎片图谱（MS2 scan）；质谱测试原始文件（Raw File）用软件Mascot2.2 检索相应的数据库（Equus caballus），最后得到蛋白质鉴定结果，结果见图11，从图中可以得到相应碎片的相应强度与荷质比，通过数据库母序列比对，确认小分子肽序列为PQPIERT，如SEQ ID NO:1所示；
（8）通过上海生工合成纯度≥98%的小分子肽PQPIERT，梯度溶解为不同浓度的小分子肽溶液，检测不同浓度小分子肽的二肽基肽酶Ⅳ抑制活性，通过计算得到小分子肽PQPIERT对二肽基肽酶Ⅳ的抑制率为86%，半抑制浓度（IC50）为0.726 mg/mL。
上述方法中半制备RP-HPLC的色谱条件：进样量1mL，流速2mL/min，检测波长215nm；流动相A含0.1%（v/v）三氟乙酸（TFA）的超纯水，流动相Ｂ为含0.1%（v/v）TFA的乙腈，洗脱条件：用流动相A和B梯度洗脱（0-5 min，10%B；5-40 min，10-50%B；40-50 min，50-80%B；50-60 min，80-10%B；60-70 min，10%B）。
实验例2：小分子肽PQPIERT对二肽基肽酶Ⅳ的酶抑制动力学实验
以0.1mol/L Tris-HCL缓冲液（pH8.0）为溶剂配制以下溶液：酶活力80U/L的DPP-Ⅳ溶液；浓度0.4、0.6、0.8、1.2、1.6和2mmol/L的GPP底物溶液；浓度范围在0-2000μm的小分子肽PQPIERT溶液，采用上述二肽基肽酶Ⅳ抑制活性检测实验方法，获得不同浓度（0、0.25、0.5mg/mL）小分子肽对不同浓度底物的吸光值变化（即反应初速度），采用Lineweaver-Burk双倒数作图，确定小分子肽PQPIERT对DPP-Ⅳ的抑制作用类型；结果见图12，从图中可以看出小分子肽PQPIERT是通过竞争性抑制作用于二肽基肽酶Ⅳ。
实验例4：小分子肽PQPIERT功能评价
（1）高糖诱导的Hepg2细胞造模
将Hepg2细胞复苏后，用含有10%FBS及1%青霉素-链霉素的高糖DMEM培养基进行培养；在37℃、5%CO2的二氧化碳培养箱培养至融合度为90%，用0.25%的胰酶进行消化，按照1:3进行传代，每2d更换一次培养基；待Hepg2细胞铺板后，弃原培养基上清，用含36 mmol/L葡萄糖的DMEM培养基诱导胰岛素抵抗模型；
阴性对照组（NT）用含5.5mmol/L葡萄糖的DMEM培养基培养Hepg2细胞；
小分子肽组用不同浓度的小分子肽（200μg/mL、100μg/mL、50μg/mL、10μg/mL）及含36mmol/L葡萄糖的DMEM进行培养；
阳性对照组（Metformin）采用36 mmol/L葡萄糖及50μmol/L二甲双胍的DMEM培养基进行培养；
模型组（IR）是采用含36mmol/L葡萄糖的DMEM培养基培养Hepg2细胞；
所有组在培养24h后收获细胞进行后续实验；
（2）CCK8细胞活性检测
将培养的Hepg2细胞用胰蛋白酶消化后，用含10%FBS的DMEM培养基制成细胞悬液，按照1.5×104加入于96孔板内，按步骤1进行分组建模，于37℃、5% CO2的细胞培养箱内培养24h，然后在各组的每孔中加入10μL CCK8试剂，在细胞培养箱中培养4h，于OD450检测吸光度，每组设置4个副孔作为对照，结果见图13，由图可知，小分子肽PQPIERT对Hepg2细胞无毒副作用且可提高细胞活性并呈剂量依赖性；
（3）葡萄糖消耗
将培养的Hepg2细胞用胰蛋白酶消化后，用含10%FBS的DMEM培养基制成细胞悬液，按照1.5×104加入于96孔板内，按步骤1进行分组建模，于37℃、5% CO2的细胞培养箱内培养24h；弃去原培养基，每孔加入100μL含100nmol/mL胰岛素的DMEM培养基于37℃、5% CO2的细胞培养箱内培养10min，弃去培养基，在各组的孔中加入100μL含11mmol/L葡萄糖的DMEM无血清培养基培养24h；采用GOD-POD葡萄糖检测试剂盒检测培养基中的葡萄糖浓度，按试剂盒操作说明进行检测；使用96孔板每孔加入3μL待测样品与300μL工作试剂（酚试剂与酶试剂按照1:1混匀），37℃孵育15min，使用酶标仪在OD 505 nm处进行测定；每组设置4个副孔作为对照，结果见图14，由图可知，小分子肽PQPIERT可显著改善高糖诱导的Hepg2胰岛素抵抗细胞葡萄糖消耗。
4、葡萄糖摄取检测
将培养的Hepg2细胞用胰蛋白酶消化后，用含10%FBS的DMEM培养基制成细胞悬液，按照2×105加入于24孔板内，按步骤1进行分组建模，于37℃、5% CO2的细胞培养箱内培养24h；弃去原培养基，每孔加入1 mL含100nmol/mL胰岛素的DMEM培养基于7℃、5% CO2的细胞培养箱内培养10min，弃去培养基，每孔加入1mL含100nmol/L的2-NBDG无血清DMEM培养基在37 ℃细胞培养箱中培养1h，1×PBS冲洗两遍停止反应，使用荧光酶标仪检测葡萄糖摄取，激发波长为475nm，发射波长为535nm，每组设置4个副孔作为对照；结果见图15，由图可知，PQPIERT可显著改善高糖诱导的Hepg2胰岛素抵抗细胞葡萄糖摄取。
序列表
<110> 昆明理工大学
<120> 马乳酪蛋白小分子肽在制备二肽基肽酶Ⅳ抑制剂中的用途
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7
<212> PRT
<213> 马乳酪(Horse cheese)
<400> 1
Pro Gln Pro Ile Glu Arg Thr
1 5
Claims (1)
1.一种马乳酪蛋白小分子肽在制备治疗2型糖尿病药物中的应用，所述马乳酪蛋白小分子肽的氨基酸序列为Pro-Gln-Pro-Ile-Glu-Arg-Thr。
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| JP2013040111A (ja) * | 2011-08-11 | 2013-02-28 | Okayama Prefecture | ジペプチジルペプチダーゼ−ｉｖ阻害剤及びその製造方法 |
| WO2013133032A1 (ja) * | 2012-03-09 | 2013-09-12 | 森永乳業株式会社 | ジペプチジルペプチダーゼ－ｉｖ阻害剤 |
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| JP2013040111A (ja) * | 2011-08-11 | 2013-02-28 | Okayama Prefecture | ジペプチジルペプチダーゼ−ｉｖ阻害剤及びその製造方法 |
| WO2013133032A1 (ja) * | 2012-03-09 | 2013-09-12 | 森永乳業株式会社 | ジペプチジルペプチダーゼ－ｉｖ阻害剤 |
| CN105273081A (zh) * | 2014-07-18 | 2016-01-27 | 中国科学院大连化学物理研究所 | 一种二肽基肽酶iv抑制肽及其制备和应用 |
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